Nanion Technologies

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Data and Applications


Accurate Pharmacology


p36_4_glycine

Two different application protocols were used to study the effect of exposure time on glycine receptor pharmacology for cells expressing hGlyRα1. As shown from the raw data traces and corresponding Hill plots, the highest concentration, 3 mM glycine, did not elicit the maximum peak response during long exposures (22 s), in contrast to stacked applications (1 s). Cells were kindly provided by AstraZeneca.

APs from SC-Derived Cardiomyocytes


AN_Patchliner_CorAtCardiomyocytes_-1

Action potentials recorded from stem-cell derived cardiomyocyetes (Cor.At® cardiomyocytes). Action potentials are triggered by small current pulses. Effects of quinidine and lidocaine on the action potentials are shown.

Cells were kindly provided by Axiogenesis.

Download: Application Note

Automated AP recordings from iCells


AP recordings Patchliner

In this example both Na+ and Ca2+ mediate the action potential. When nifedipine is applied in the current clamp mode, the action potential is shortened significantly due to block of the calcium channels.
The stem cell-derived cardiomyocytes (iCell) were kindly supplied by CDI.

Bilayer Recordings on the Patchliner


p37_2_BilayRec

With suction the GUVs are attracted to the aperture. As soon as one GUV hits the glass substrate, it bursts and forms a bilayer across the aperture. Shown are single channel recordings from gramicidin which was incorprated into the bilayer after its formation. Traces were recorded in 100 mM HCl at −100 mV.

BK - Activation by Internal Calcium


BK_vertical

Top: BK (KCa1.1) current voltage relationships in a single cell showing effects of changing the intracellular free Ca2+ concentration (15 nM, n = 9; 108 nM, n = 11; 316 nM, n = 11). Bottom: Comparison of BK (KCa1.1) current voltage relationships obtained on a conventional patch clamp setup (closed circles, n = 10) and on the Patchliner® (open circles, n = 11).
Data are taken from Milligan C.J. et al., Nature Protocols, 2009, 4(2), 244-255.

Cav2.2 Cadmium Block


Cav2.2 Cadmium Block

The image shows current response of an individual cell in the presence of increasing cadmium concentrations. The IC50 was calculated from the Hill fit to be 3.6 ± 0.4 μM (n = 5).

Cells were kindly provided by Millipore.

Cav2.2 Current-to-Voltage Relatiotionship


Cav2.2 I/V-Curve

Representative current responses of an individual cell expressing Cav2.2 to a I/V voltage protocol. The average peak current at 30 mV of all recorded cells was -698 ± 115 pA (n = 6).

Cells were kindly provided by Millipore

Cav3.2 Current-to-Voltage Relationship


IV_Cav32

Representative current responses of an individual cell expressing Ca 2.2 to a standard voltage protocol. The average mean current at -20 mV of all recorded cells was -785 ± 110 pA (n = 12).
Cells were kindly provided by Millipore


Cav3.2 Inactivation


Cav32_inactivation

Current responses of a double pulse protocol with varying test potentials between the pulses (5 s) was used to determine the half inactivating potential. Peak current responses to the second pulse are expressed relative to the response to the first pulse. Both curves in Figure 5 were fitted to the Boltzmann equation and revealed a half-inactivating potential of -65 mV and a half-activating potential of -33 mV.

Cav3.2 Mibefradil Antagonism


Cav32_Mibefradil

Dose dependent block by Mibefradil on current traces from an individual cell expressing Ca 3.2. Data was averaged and fitted to the Hill equation where the IC50 value was determined as 1.7 µM. Error bars corresponds to S.E.M.
Cells were kindly provided by Millipore.

CFTR Regulation


CFTR

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is activated by forskolin. The upper graph shows the timecourse of currents recorded at +95 mV. The bar above the data indicates the time of compound application. Arrows indicate data from which time points were averaged in the lower figure (n = 3).

Here you can find the full report.

Currents from SC-Derived Cardiomyocytes


p34_4_actPot

The left picture shows a typical action potential from Cor.At® cardiomyocytes. Whole cell currents recorded in the voltage clamp mode reveal cardiomyocyte-typical ion channels (right). The traces represent mERG-, L-type Ca2+- (blue, block by 50 μM nifedipine), Na+- and K+-currents (from top left to bottom right).

Cells were kindly provided by Axiogenesis.

Efficient hERG Screening


p34_2_hERG_II_Sum

The effects of six different blockers (terfenadine, cisapride, E4031, astemizole, propafenone, quinidine) on hERG currents (HEK293 cells) were investigated. Expected IC50 values for the different compounds were obtained. In two days, 119 full dose response curves were collected by a single person. Data was analyzed using Nanion’s Data Analysis Package, a very efficient and convenient data analysis tool!

Cells were kindly provided by Cytomyx/Millipore, UK.

Erythrocytes - Single Channel Recordings


application16_erythro_1-small

The membrane of erythrocytes contains different ion channels like Ca2+-activated K+ channels, or the volume-sensitive Na+/K+ pump. Studies also revealed the participation of a Ca2+-permeable non-selective cation channel in the regulation of erythrocyte 'apoptosis'. Shown are single channel fluctuations as recorded from an erythrocyte in the cell attached configuration on the Patchliner®.

Erythrocytes - Whole Cell Recordings


PatchlinerRBC

Whole cell current recordings from erythrocytes recorded on an eight-channel Patchliner®.

Cellswere kindly donated by Dr. Andrea Brüggemann.

GABAA Receptor - Activation


application16_gabaa_1_copy

Shown is concentration dependent activation of GABAA (α1β2γ2) receptors by 1, 3 and 10 μM GABA from a GABAA (α1β2γ2) expressing HEK293 cell. The rising phase in enlarged in the lower graph.

GluR2 Receptor - Fast Activation


GluR2 CRC  current time course

Shown is concentration dependent activation of GluR2 receptors (known as AMPA receptors) by 10, 30, 100 μM and 300 µM Na-Glutamate from a GluR2 expressing HEK293 cell. The rising phase is enlarged in the right graph. Cells were kindly provided by University of Sussex.

Glycine Receptor - Antagonist


application16_glycine_3

The figure shows current responses of a hGlyRα1 expressing L-tk cell to alternating exposures to 100 μM glycine and 100 μM glycine + 1 μM strychnine.

Cells were kindly provided by Astrazeneca, Södertälje, Sweden.

Glycine Receptor - Dose Response Analysis


PLGlycineLynch

(A) Effects of indicated concentrations of 3 on α1 and α3 GlyR currents activated by EC20 gycine concentrations as indicated. Unfilled bars denote glycine applications and filled bars denote compound applications. (B & C) Average dose response curves of 3 at α1 and α3 GlyRs, respectively.
Data are taken from Balansa W. et al., Bioorg Med Chem. 2010 Apr 15;18(8):2912-9.

Glycine Receptor - Potentiation


application16_glycine_2

Original traces of one application of 20 μM glycine followed by 6 applications of 20 μM glycine in conjunction with increasing concentrations of a positive modulator. 1 mM glycine was used as a second positive control.

Cells and the positive modulator were kindly provided by Astrazeneca, Södertälje, Sweden.

 

hERG - Stable Recordings


application16_herg_1-small

A series of drug concentrations can be applied to each cell. The top figures show the original traces and the corresponding average dose-response curve. Five concentrations of Quinidine (0.1, 0.3, 1, 3 and 10 μM) have been applied.

The lower figure shows the corresponding Imax (-40 mV) including a wash out step and an additional application of the blocker to demonstrate the stability of whole cell recordings.

Cells were kindly provided by Cytomyx/Millipore, UK.


hERG - Sticky Compounds


application16_herg_2

Even sticky compounds pose no problem for the Patchliner®. IC50 measurements of well known sticky substances were determined on the Patchliner®: Terfenadine IC50 = 11.0 ± 3 nM, Flunarizine IC50 163.7 ± 19 nM and Cisapride IC50 8.9 ± 3 nM.

hERG expressing HEK293 cells were kindly provided by Cytomyx/Millipore, UK.

hERG Block at Physiological Temperature


AppNote_hERG_TEmp

The effects of erythromycin on hERG currents were tested at different temperatures. Erythromycin has been shown to block hERG channels at physiological temperature with an IC50 of approx. 40 µM. However, at RT erythromycin is much less potent. For more details on these experiments please refer to the Application Note. Cells were kindly provided by Cytomyx/Millipore, UK.

hERG- Easy Data Analysis


hERG_Screenshot_Data_Analysis_Tool_1_700

We have great data analysis tools! - Especially programmed routines in Igor make dose response curves, raw data and current time courses easily accessible. Also, creating average dose response curves over multiple experiments - even conducted on different days - remains easy.


hNav1.5 - Lidocaine Block


application_hnav15_3-small

Full dose response curves at different holding potentials were recorded for each cell (hNav1.5 in HEK293). Currents were elicited by a 10 ms voltage step to 0 mV. Plotted are average peak currents as a function of holding potential and lidocaine concentration. Cells were kindly provided by Cytomyx/Millipore, UK.

iCell Neurons - GABA Currents


GABA_Bicuculline_neurons

GABA currents in iCell Neurons. Activation of GABA currents by 30 μM GABA and partial block of the GABA response by 1 μM bicuculline. Bicuculline was pre-applied for at least 30 s before co-application with GABA (30 μM). Approximately 50% of the current was blocked by 1 μM bicuculline.

Cells were kindly provided by CDI.

iCell Neurons - TXX Block of Na-Currents


TTX_Neurons_Figure2

A Block of Na+ current by increasing concentrations of TTX. Current was blocked by low nM concentrations of TTX indicating a TTX sensitive Na+ channel type expressed in this cell. B Concentration response curve for TTX inhibition, IC50 = 4.9 nM (n =1).

Cells were kindly provided by CDI.

iCell Neurons - Voltage-Gated K-Currents


KvIV_Neurons_Figure3

A Voltage-gated K+ current recorded in iCell® neurons. Current responses to a voltage step protocol. An outward K+ current can be seen in this cell. B Corresponding IV plot from an average of 7 cells.

Cells were kindly provided by CDI.

iCell Neurons - Voltage-Gated Na-Currents


NaIV_Neurons_Figure1

Voltage-gated Na+ current recorded in iCell® Neurons. Current responses to a voltage step protocol. A large inward Na+ current can be seen in this cell with a K+ outward current present at positive voltages.

B Normalised IV plot from an average of 4 cells.

Cells were kindly provided by CDI.

Internal Perfusion on Primary Rat Astrocytes


p35_3_ratAstrocytes_2

Whole cell currents from rat cortical astrocytes (primary culture) were evoked by 500 ms long depolarizing voltage steps (-100 mV to +40 mV). Currents were blocked by administration of internal Cs+, and recovered when switching back to Cs+-free internal solution. Averaged data are presented as mean ± S.E.M. (n=35). For more information, see Nature 254, 4 (2), 2009. Data courtesy of C. Peers, University of Leeds, Leeds, UK.

Internal Solution Exchange


p35_4_ExchInternSol

A unique feature of the Patchliner® is its ability to exchange the internal solution during the recording. The figure shows recordings of Kv1.3 from two Jurkat cells (simultaneoulsy recorded) in the presence of control internal solution, after the exchange of the internal solution with a Cs+ solution, and subsequent washout (left to right).

Investigation of Modulators


p37_1_modulators

The top images show dose dependent block of GABAA currents by bicuculline. The IC50 was determined as 1.2 ± 0.2 μM (n=11). The lower graph shows the positive modulation of glycine activation of hGlyRα1. Here, six co-applications of 20 μM glycine and increasing concentrations of a positive modulator are shown. Cells were kindly provided by AstraZeneca.

Inward Rectifier - Kir2


application16_kir2_2-small

The basophilic leucaemia cells (RBL) exhibit an inwardly rectifying potassium current, Kir. Changing the external K+ concentration (here between 4.5 mM and 143 mM) leads to a change of the current amplitude of the inward current (holding: -100 mV). This gives us a convenient tool to study the speed of the external solution exchange which was determined as ~50 ms.

KCNQ1 Current Voltage Recordings


PL_KCNQ1

Screenshot of KCNQ1 recordings obtained on an eight-channel Patchliner®. Currents are responses to an current voltage relationship type step pulse protocol.

Kv Currents in Astrocytes


astrocytes

Left: Comparison of K+ current voltage relationships for rat astrocytes on the Patchliner (closed circles, n = 19) and on a conventional setup (open squares, n = 10). Currents were measured as a response to a voltage step protocol. Right: Normalized K+ current amplitudes in rat astrocytes. Internal solution was changed to the same solution (K+, open circles, n = 7) or to one where Cs+ was substitued for K+ (Cs+,closed circles, n = 7). Currents were measured as responses to voltage steps from −100 mV to +40 mV.
Data are taken from Milligan C.J. et al., Nature Protocols, 2009, 4(2), 244-255.

Kv Currents in Lymphoblasts


Lymphoblast

An illustrative typical family of traces obtained during construction of a current voltage relationship from a human lymphpblast recording using amphotericin B to access th whole cell. Currents were evoked by voltage steps from holding (−80 mV) to potentials varying from −100 mV to +60 mV.
Data are taken from Milligan C.J. et al., Nature Protocols, 2009, 4(2), 244-255.

Nav Currents in Cor.At® Cardiomyocytes


p35_1_CorAt

The pharmacology of dibucaine was investigated by the application of 0.3, 1, 3, 10 μM in the presence of 10 μM nifedipine (L-type Ca2+-current blocker). Two control additions of nifedipine (10 μM) were made before the addition of increasing concentrations of dibucaine. The IC50 value was determined as 355 ± 40 nM (n=3), which is in accordance with the literature. Cells were kindly provided by Axiogenesis.

Nav1.8 Block by Tetracaine


ND723_Nav18_Tet_Figure

A Raw traces from an exemplar cell recorded on the Patchliner showing inhibition of current by increasing concentrations of tetracaine. Shown are current responses to a single step protocol to 20 mV for 25 ms from a holding potential of -90 mV. Current amplitude was completely recovered upon washout of tetracaine (red trace). B Timeplot of the experiment. Cells were kindly provided by Millipore.

Nav1.8 I/V Characteristics


ND723_PL_Nav18_IV_Figure

A Raw traces from an exemplar cell recorded on the Patchliner. Shown are current responses to increasing voltage steps from -80 to +60 mV. B Average current-voltage plot, Vhalf of activation was -9 mV (n = 19). C Average inactivation plot, Vhalf of inactivation was -24 mV (n = 4). Nav1.8 currents started to activate at about -40 mV, peak response was elicited at around 20 mV

Cells were kindly provided by Millipore.

Nav1.8 Voltage Dependent Block by Tetracaine


IC50_TetracaineOverlay

Recordings were made on the Pachliner. The potency of tetracaine was affected by holding potential, becoming less potent with a more negative holding potential. Average concentration response curve for tetracaine, IC50 = 35 ± 8 μM (n = 3) for Vhold - 90 mV and 74 ± 15 μM (n = 4) for Vhold - 120 mV.

Cells were kindly provided by Millipore.

Nicotinic AChR a7 Dose response curve


PL_Nica7_DR

Complete nicotine dose response curves were obtained by applying increasing concentrations of nicotine to a HEK293 cell expressing human nicotinic α7 acetyl choline receptors. The stacked application protocol was used.

Cells were kindly supplied by Galantos Pharma GmbH.

Nicotinic AChR a7 Stable nicotine responses


Repeated nicotine responses

Stable whole-cell current amplitudes were obtained by repeated nicotine stimulation of HEK293 cells expressing human nAChR a7 receptors. The stacked application protocol was used. Cells were kindly supplied by Galantos Pharma GmbH.

P2X7


P2X7

BzATP concentration response curve obtained from wild-type P2X7 receptors. Data are fite to the Hill equation with an EC50 of 69.7 ± 7.5 μM. The inset show representative currents evoked by BzATP (3 − 100 μM). Holding potential was −60 mV.

Data are taken from Milligan C.J. et al., Nature Protocols, 2009, 4(2), 244-255.

Reproducible Compound Application


p36_1_reproducRec

Application of 5 μM quinidine leads to about 50 % block of the Kv1.3 currents (blue). After washout, the current is fully recovered (grey). The lower graph shows corresponding Imax (+40 mV) in the absence and presence of 5 μM quinidine, for two different cells with eight consecutive application and washout steps. The recording lasted over 40 minutes!

Stable Access Resistance


p35_2_VoltContr

The I/V-characteristics of Nav1.5 currents (HEK293) are shown together with the repeated dose dependent block by TTX (lower panel). Five concentrations of TTX
(0.3, 1, 3, 10, 30 μM) were applied, followed by washout with antagonist-free buffer and re-application of the same TTX concentrations. Cells were kindly provided by Millipore.

Stacked Applications - Brief Exposures


p36_3_timedInterv

Ligand gated ion channels often display receptor desensitization. A method was developed to minimize ligand exposure times and intervals between ligand exposures. The pipette first aspirates buffer, then compound. When expelling this stack, the cell is first exposed to ligand and then buffer. Exposure times as low as 400 ms are possible with this method. A GABA dose response curve, aquired in this manner, is shown on the left.

Transient Heat Activation of TRPV1


p36_2_TRPV1

The current responses of a CHO cell expressing TRPV1 (ramp -100 mV to +100 mV) at increasing temperatures is shown. The ET50 value was determined as 64°C. Challenge of the same cell with capsaicin (1 μM) and temperature (70°C) allows comparison of the responses. IC50s for ruthenium red block of capsaicin- and heat-responses were determined as 1.6 ± 0.2 μM (n = 3) and 7.4 ± 1.3 μM (n = 3), respectively.

TRP A1


TRPA1_Screenshot

Shown is a screenshot from a recording on a 4-channel Patchliner from HEK293 cells expressing TRP A1. Currents were activated by ca. 20 s application of 3 μM AITC followed by wash out. On the left raw whole cell currents as responses to 0.2 s voltage ramps (−100 mV to +100 mV) which were applied every 10 s are shown. On the right currents at +95 mV are plotted against time. (The time course of a similar experiment is shown here.)

Cells were kindly supplied by Millipore.

TRP A1 Current Time Course


TRPA1_Patchliner

Shown is the time course of the current measured at +95 mV from an individual cell (these are data from a similar experiment as shown in TRP A1). The legend indicates the time periods in which the activator AITC was applied. Here we demonstrate the reproducibility of the current responses for TRP A1.

Cells were kindly supplied by Millipore.

TRP C5


TRPC5

Current voltage relationships showing activaion of TRPC5 by extracellular application of Gd3+ (100 μM). Voltage ramps (−100 mV to 100 mV, holding potential 0 mV) were for 1s at 0.1 Hz.

Data are taken from Milligan C.J. et al., Nature Protocols, 2009, 4(2), 244-255.

TRP Currents in Smooth Muscle Cells


smoothMuscle

Left: Illustrative time series showing currents at +80 mV and −80 mV in a smooth muscle cell exposed to extracellular Gd3+ (100 μM) and then 2-APB (75 μM). Right: Membrane resistance (Rm), series resistance (RS) and membrane capacitacne (Cm) values for successful recordings from smooth muscle cells.
Data are taken from Milligan C.J. et al., Nature Protocols, 2009, 4(2), 244-255.

TRP M3


PL_TRPM3

Shown are whole cell currents as responses to 0.2 s voltage ramps (−100 mV to +100 mV) which were applied every 10 s. All cells were HEK293 cells induced to express TRPM3. (a and c) Mean currents sampled at −80 and +80 mV, each normalized to the amplitude immediately before bath application of antiserum (TM3E3; 1:500 dilution) without (a, n = 8) or with (c, n = 4) pre-adsorption to 10 μM antigenic peptide. Pregnenolone sulphate
(PregS) was bath applied at 25 μM. (b and d) Typical current voltage relationships from the experiments underlying (a) and (c).
Data are taken from Naylor J. et al., British Journal of Pharmacology, 2008, 1-7.

TRPC1 Currents in Human Saphenous Vein


PL_TRP_SMC

(a) Example whole cell recording showing current evoked by extracellular applicaiton of 2 μM thapsigargin (TG) and subsequent inhibition by 10 μM lanthanum (La3+). (b) As for (a) but showing the effect of 20 μg/ml anti-STIM1 antibody. (c) For the experiment shown in (b), curren voltage relationships for the current evoked by TG and blocked by anti-STIM1 antibody. (d) Mean currents normalized to pre-antibody values showing the effect of 20 μg/ml ani-STIM1 antibody and lack of effects for IgG or anti-STIM1 anibody denatured by boiling.
Data are taken from Li, J. et al., Circulation Research, 2008,103(8), e97-104.

TRPM3 positive modulation


TRPM3_PS_C_Data

A Current responses to a voltage ramp protocol from -150 mV to 150 mV over 200 ms in response to 100 μM pregnenolone sulphate (PS) and in combination with increasing concentrations of compound C. B Current responses elicited by PS, enhancement by 30 μM compound C and block of the current by co-application of antagonist (10 μM). Cells were kindly provided by Prof. Thomas Voets, KU Leuven, Belgium.

TRPV3 Temperature Activation


TRPV3_Patchliner

Recordings from a HEK cell expressing TRPV3 were made with the Patchliner showing activation by heat. External solution was heated inside the Patchliner pipette to the temperature shown and applied to the cells. TRPV3 was activated at temperatures ≥ 38°C.

Cells were kindly supplied by Millipore.